Infertility is a major public health problem that affects 10-15% of couples (2.5 million) in the US. The goal of this program is to locate genes that make substantial contributions to variation in natural dizygotic (DZ) twinning to understand critical pathways controlling female fertility. This will have practical application in treatment of infertility and may provide new approaches to contraception. The proposed study extends our previous studies of the endocrinology, epidemiology, and molecular genetics of twinning in humans and in large animal models. We have ascertained 342 families with sister pairs who have both given birth to spontaneous DZ twins and a further 386 families with case-two parent triads and have collected DNA samples from the sisters' parents and other siblings. We shall use information from our twin studies and the Australian and Netherlands Twin Registries to collect an additional 240 affected sister pair families from Australia and New Zealand and 418 families from the Netherlands. DNA samples from these families will be used to complete a 10cM genome scan in 1000 families. We will use sib pair linkage analysis to locate QTLs for twinning. Our candidate positional cloning in animal models recently identified mutations in two genes (BMP15 and BMPRIB) from intra-ovarian signaling pathways that increase twinning frequency, and cause infertility in homozygous carriers of the BMP15 mutations. One important question is whether natural variation in human twinning is controlled by these emerging mechanisms or through other pathways. We will use available samples to immediately begin candidate gene studies by mutational screening and typing SNP markers in genes from this pathway (BMP15, GDF9, BMPR1B) using a matched case-control design with cases from our existing families and matched control samples. Significant associations will be tested in additional cases and their parents using the transmission disequilibrium test.